Myosin is a family of proteins that demonstrate force-generated ATPase activity when it interacts with actin. Myosin heavy chain (MHC) protein isoform diversity in vertebrates is generated by multiple genes as well as by alternative splicing of the pre-mRNA. Previous studies in this laboratory have demonstrated the existence of a neural cell-specific nonmuscle MHC-B isoform, in addition to the ubiquitously expressed form of nonmuscle MHC-B. This neural cell-specific isoform is generated by alternative splicing of a single cassette type of exon N30 which is located between constitutive exons E5 and E6. Inclusion or exclusion of exon N30 is cell type-specific, with inclusion being restricted to neural cells and being regulated during cell differentiation. We have recently identified an intronic distal downstream enhancer (IDDE) region which is required for neural cell-specific inclusion of N30. We have been investigating the mechanism by which the IDDE promotes N30 exon usage. In vitro splicing analysis using neural cell nuclear extracts and two-exon pre-mRNA substrates, which consist of the N30 exon and either the upstream (E5) or downstream (E6) exon, demonstrates that the IDDE activates upstream E5-N30 splicing by facilitating early prespliceosome complex formation. The IDDE has no effect on N30-E6 splicing where the IDDE resides. Inspection of splice site consensus sequences shows that a polypyrimidine (Py) tract preceding N30 is suboptimal for U2AF binding. Optimizing the Py tract completely relieves the requirement for the IDDE in E5-N30 splicing in vitro. In transfected cells, the wild-type minigene transcripts, which consist of three exons, E5, N30 and E6, undergo neural cell-specific and IDDE-dependent alternative splicing of N30. Optimizing the Py tract in minigenes also completely relieves the requirement for the IDDE in N30 inclusion. Furthermore, overexpression of the truncated U2AF65, which contains the arginine and serine-rich domain and linker domain, but lacks the RNA binding domain, selectively inhibits the IDDE-mediated N30 inclusion in mRNA from the wild-type minigene in a dominant negative fashion. These results support the hypothesis that the IDDE facilitates the recognition of the 3' splice site preceding N30 by a network of protein-protein interactions implicated in the recruitment of U2AF to a suboptimal Py tract.